CN110054665B - 一种环五肽化合物在制备免疫抑制剂药物中的应用 - Google Patents
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Abstract
本发明属于药物技术领域,具体是涉及到一种从猴头菌丝体中分离的环五肽化合物、制备方法和其在制备免疫抑制剂药物中的应用,其结构如下:
Description
技术领域
本发明属于药物技术领域,具体是涉及到一种从猴头菌丝体中分离的环五肽化合物、制备方法和其在制备免疫抑制剂药物中的应用。
背景技术
猴头菌丝体原名猴头菌培养物, 现行标准收载于《中国药典》2015 年版一部猴头健胃灵片制剂项下,是猴头健胃灵片和猴头健胃灵胶囊的主要成分。目前有关猴头菌丝体的文献报道主要集中在其多糖方面,研究表明,猴头菌多糖具有多种生物活性和药理作用,能增强巨噬细胞的吞噬功能,促进溶血素的形成,抗白细胞下降,降血糖、抗凝血、抗血栓、抗突变和抗衰老等。
猴头菌丝体含有多种成分,包括多糖、多肽、活性纤维素酶、镁、锌和硒等,如何将猴头菌丝体中的有效成分进行提取和纯化,成为一个比较重要的研究课题。
中国专利申请号为201810257329.4的中国专利公开了一种从猴头菌丝体中分离的化合物,为有机化合物,其主要骨架为苯环等基团,主要用于抗肿瘤。
发明内容
本发明要解决的技术问题是提供一种从猴头菌丝体中分离的环五肽化合物、制备方法和其在制备免疫抑制剂药物中的应用。
本发明的环五肽化合物,结构如下:
所述环五肽化合物的制备方法,包括如下步骤,
1、将猴头菌丝体经水提后再减压浓缩,得到猴头菌丝体水提物,将猴头菌丝体水提物加水制成悬混液,用乙酸乙酯萃取,减压浓缩去除乙酸乙酯,得到乙酸乙酯层浸膏,将浸膏和硅胶混合,用硅胶填充色谱柱,上样,采用二氯甲烷和甲醇混合液进行梯度洗脱,将洗脱下来的流份浓缩,合并具有相同RF值和相同斑点颜色的流份,按照先后顺序得到10个组分;
2、将第6个组分用氯仿溶解,将其和硅胶混合,用硅胶填充色谱柱,上样,采用石油醚和丙酮混合液进行梯度洗脱,将洗脱下来的流份浓缩,合并具有相同RF值和相同斑点颜色的流份,按照先后顺序得到8个组分;
3、将第4个组分用氯仿和甲醇溶液溶解,过滤,采用葡聚糖凝胶柱色谱,用氯仿和甲醇混合液进行洗脱,薄层色谱检测蓝色和灰色主斑点的成分并洗脱,剩余部分用水溶解,过滤,用反向C18色谱柱以甲醇和水混合液进行洗脱,得到环五肽化合物。
优选的,所述步骤3中的过滤的滤膜孔径为0.45微米。
本发明还提供一种环五肽化合物在制备免疫抑制剂药物中的应用。
本发明的免疫抑制剂药物的剂型包括胶囊、片剂、针剂、口服液、注射用冻干剂或粉针剂等。其剂量一般在0.01~5mg/kg体重,优选0.5~3mg/kg体重,可以一次或多次施用。
本发明的有益效果是,本发明采用从猴头菌丝体中提取得到,分子量较少,表现出良好的低毒性和对淋巴细胞增殖的抑制作用,具有优异的免疫抑制能力。
具体实施方式
实施例1
1、猴头菌丝体水提取物的萃取
猴头菌丝体水提取液浓缩,得25kg猴头菌丝体水提物,将猴头菌丝体水提物加水制成混悬液,用等量乙酸乙酯萃取混悬液重复3次。合并有机层,得到乙酸乙酯萃取液,用旋转蒸发仪减压浓缩,蒸干乙酸乙酯得到乙酸乙酯层浸膏。
2、猴头菌丝体乙酸乙酯部分分离
乙酸乙酯层浸膏共63.5g,用1.5倍浸膏量的硅胶(180-100目)100g拌样,使其均匀吸附在硅胶颗粒表面,用50倍浸膏量的硅胶(100-200目)3000g填装色谱柱,干法上样,按照二氯甲烷:甲醇=(100:1,80:1,60:1,40:1,20:1,10:1,5:1,3:1,1:1,1:5)梯度洗脱,500ml/流份,将洗脱下来的流份浓缩,TLC检验(硅胶GF254板,显色剂为5%香草醛-浓硫酸)合并具有相同Rf及斑点颜色相同的流份,按照流出的先后顺序共得10个组分Fr A-J。
Fr F(I2.8g)用氯仿溶解,1.5倍浸膏量的硅胶(80-100目)16.5g拌样,用50倍浸膏量的硅胶(200-300目)600g填装色谱柱,按照石油醚:丙酮=(60:1,40:1,20:1,10:1,5:1,3:1,1:1)梯度洗脱,200ml/流份,将洗脱下来的流份浓缩,TLC检验(硅胶GF254板,显色剂为5%香草醛-浓硫酸)合并具有相同Rf及斑点颜色相同的流份。共得8个组分Fr I-VII。
Fr F.IV用氯仿:甲醇=(0:1)溶解,0.45μm微孔滤膜过滤,湿法上样,反复通过葡聚糖凝胶Sephadex LH-20柱色谱,用氯仿:甲醇=(1:1)洗脱得到化合物(3.5mg),将TLC检识具有蓝色主斑点的成分继续用半制备型高效液相色谱分离,通过30%甲醇-水等度洗脱得到化合物(4.1mg),通过35%甲醇-水等度洗脱得化合物(5.6mg),将TLC检识具有灰色主斑点的成分继续用半制备型高效液相色谱分离,通过35%甲醇-水等度洗脱得到化合物(4.0mg),将剩余部分用水溶解,0.45μm微孔滤膜过滤,湿法上样,用反相C18色谱柱甲醇:水=(0:100-100:0)洗脱得到本发明化合物(7.8mg)。
3、猴头菌丝体化学成分的结构鉴定
所得单体化合物在美国密西西比大学及湖南中医药大学进行了1H-NMR,13C-NMR,HMBC,HSQC,1H1H COSY测试。经过谱图解析得到单体化合物结构。
本发明化合物名称为:环(亮氨酸-亮氨酸-缬氨酸-亮氨酸-异亮氨酸)。为白色无定型粉末(氯仿:甲醇=1:1),5%香草醛-浓硫酸TLC显色反应为深褐色。
HR-ESI-MS谱中给出[M+Na]+峰574.3940(计算值574.3944).可推断化合物分子式为C29H53O5N5。
本发明化合物的13C-NMR(125MHz)谱显示有重叠碳信号,查阅文献,对比氨基酸碳化学位移,找出3个氨基酸单元分别为缬氨酸(Valine),异亮氨酸(Isoleucine),亮氨酸(Leucine)。结合1H-NMR(500MHz)谱和HSQC谱推测缬氨酸碳化学位移为δc16.1(CH3)、18.1(CH3)、32.1(CH)、60.0(CH)、168.6(C=O)。异亮氨酸碳化学位移为δc11.4(CH3)、14.7(CH3)、24.0(CH2)、39.0(CH)、59.6(CH)、168.7(C=O)。亮氨酸碳化学位移为δc21.4(CH3)、22.7(CH3)、24.0(CH2)、42.0(CH)、53.0(CH)、169.8(C=O)。此外根据1H-NMR(500MHz)谱积分推测3个亮氨酸信号重叠。
通过HMBC谱,H-1对C-2/3/4/5有相关,H-2对C-1/3/4有相关证明该片段为缬氨酸,H-1'对C-3'/4'/5'有相关,H-3'对C-1'/2'/4'/5'有相关证明该片段为异亮氨酸,H-1”对C-2”/3"/6”有相关,H-2”对C-1”/2”/3”/4"/5”/6''有相关证明该片段为亮氨酸。此外,由于H-1与C-6”有相关,推测缬氨酸与亮氨酸相连接,H-1'与(C-6”、H-1”与C-5/5'6”有相关,推测异亮氨酸氨酸与亮氨酸相连接,故该化合物结构为环五肽。经结构鉴定,该化合物命名为环(亮氨酸-亮氨酸-缬氨酸-亮氨酸-异亮氨酸)。本发明化合物的1H和13C的NMR数据如表1所述。
表1 本发明化合物的1H和13C的NMR数据(MeOD/CDCl3)
实施例2
本发明的环五肽化合物在刀豆蛋白诱导的活化T细胞增殖实验上显示抑制活性。实验过程和结果如下:
正常机体的T淋巴细胞在体外培养过程中,受到特异性抗原或有丝分裂原刺激,细胞的代谢和形态可发生一系列变化,主要表现为电荷的改变,细胞体积增大,代谢旺盛,细胞内蛋白质和核酸合成增加(并能进行分裂),以及细胞形态可转化为淋巴母细胞,即为淋巴细胞转化现象。为此,在体外利用各种刺激剂激发淋巴细胞,根据其转化程度可测定T淋巴细胞的应答功能(淋巴细胞转化率的高低可以反映机体细胞免疫水平)。本实验应用刀豆蛋白(Con A)刺激T淋巴细胞,然后用MTT法测定细胞存活量。
实验过程:取5×105cells/well的C57BL/6小鼠淋巴结细胞置于96孔培养板中,药物组与Control组加入终浓度为5μg/ml的Con A溶液,置37℃,5% CO2培养箱中,培养1天。1天后采用MTT测细胞存活率。记录结果,以浓度为横坐标,T细胞存活率为纵坐标绘制曲线,计算化合物的IC50值,其IC50值为10μM。
本发明化合物对活化T细胞增殖显示出一定的抑制作用。抑制率接近环孢菌素(CsA),具备成为新的环肽类免疫抑制剂的前景。
实施例3
采用6-7周小鼠作为实验对象,18-20g,雌雄各半。
断颈处死小鼠,无菌取脾,置于盛有适量无菌Hank’s液平皿中,用镊子轻轻将脾磨碎,制成单个细胞悬液。经200目筛网过滤,或用4层纱布将脾磨碎,用Hank’s液洗2次,每次离心10min(1000r/min)。然后将细胞悬浮于1mL的完全培养液中,用台酚兰染色计数活细胞数(应在95%以上),调整细胞浓度为3×106个/mL。
将脾淋巴细胞悬液接种到96孔培养板中,每孔100μl,设6个复孔。空白对照组:每孔加100μl的RPMI1640培养基;阳性对照组:每孔加100μl环孢素(500ug/ml,用无水乙醇配制成5mg/mL的溶液,过滤除菌,在低温冰箱-20℃保存,用时用完全培养液稀释到所需浓度);多肽组:每种多肽(以无水乙醇为溶剂,将多肽配制成5mg/ml的溶液;然后用完全培养液稀释到我们所需要的浓度)分为高(500ug/ml)、低(50ug/ml)两个试验组,每孔加100μl。
将96孔板置CO2培养箱中(5%CO2,37℃)培养48h。培养结束后,每孔添加20ul的MTT(5ug/ml),在5%CO2,37℃条件下培养4h,离心(1000r/min×5min),吸弃上清,每孔添加二甲亚砜150ul,轻轻震荡10min以溶解紫色结晶沉淀,测定在492nm波长处的光吸收值。
将实验组和对照组6个复孔的OD各自取平均值,用下式计算抑制率,结果如表2所示。
表2环五肽化合物对淋巴细胞增殖的影响
组别 | 抑制率 |
空白对照 | - |
阳性对照 | 12.5% |
高剂量组(500ug/ml) | 12.9% |
低剂量组(50ug/ml) | 10.1% |
利用MTT法测得的环孢菌素(阳性对照)组对小鼠淋巴细胞的抑制率为12.5%,本发明环五肽的高、低剂量组的抑制率分别为12.9%和10.1%,与环孢菌素水平相当,表明其具有优良的免疫抑制能力。
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